Heteroaryl-containing isoflavones as aromatase inhibitors

ABSTRACT

Compounds and methods useful for treating and prevention of cancer, particularly hormone-dependent breast cancer. Provided are compounds of formula I: 
                         
wherein X is selected from O, N, S, SO, SO 2 , and S(CH 2 ) n , wherein n=1-10; R 1  and R 2  may be the same or different and are selected from H, OH, OCH 3 , OCH 2 CH 3 , OCH 2 C 6 H 5 , NH 2 , NHCH 3 , N(CH 3 ) 2 , CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 , CH(CH 3 ) 2 , C(CH 3 ) 3 , NO 2 , F, Cl, Br, CF 3 , SH, SCH 3 , SCH 2 CH 3 , OCOCH 3 , OCOC(CH 3 ) 3 , OCOCH 2 COOH, and CN; and R 3  is a nitrogen-containing heterocyclic ring. Also provided are method for treating or preventing cancer in a subject by administering a therapeutically effective amount of a heteroaryl-containing isoflavone, or a pharmaceutically acceptable salt or prodrug thereof, to a subject in need of treatment. Also provided is a method for the synthesis of 2-substituted isoflavones by first reacting deoxybenzoins with a phase transfer catalyst to provide a 2-(alkylthio)isoflavone; second deprotecting the 2-(alkylthio)isoflavone; and third applying selective debenzylation to form the final compound.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims priority toU.S. patent application Ser. No. 11/212,229, filed on Aug. 26, 2005 nowU.S. Pat. No. 7,572,919, titled “HETEROARYL-CONTAINING ISOFLAVONES ASAROMATASE INHIBITORS”, which in turn claims priority to U.S. ProvisionalPatent Application No. 60/604,667, filed Aug. 26, 2004, both of whichare hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was supported at least in part by US Army MedicalResearch and Material Command Grants DAMD 17-99-1-9342 and DMAD17-00-1-0388. The Federal Government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Flavonoids are a diverse group of plant-derived chemicals that areproduced by various higher plants, which can be found in numerous foodsources such as fruits, vegetables, legumes, and whole grains. Compoundsin this class have shown a wide variety of biological activities such asanti-inflammatory, antibacterial, antifungal and anticancer activities.In particular, due to their structural and functional similarities toendogenous estrogens, flavonoids have attracted considerable interest asalternative estrogens, termed phytoestrogens, and extensively studiedfor their potential role in many estrogen-dependent diseases includingbreast cancer. In fact, numerous flavonoids have shown interestingpharmacological activities and inhibitory activities against aromataseenzyme.

Genistein (GEN) is the most abundant isoflavone in soybeans. Someresearchers suggest that GEN may be responsible for the relatively lowincidence of hormone-dependent breast cancer in certain regions withhigh consumption of soy foods. Indeed, GEN has been one of the mostwidely studied natural products and has shown a number of importantbiological activities in breast cancer biology. GEN displays moderatebinding to estrogen receptors, as well as exhibiting antiproliferativeactivity in many can cell lines mediated by several mechanisms ofaction. Furthermore, it is known that GEN exerts many beneficial effectson endogenous estrogens on several tissues such as bone andcardiovascular system. In addition to estrogenic activities, GEN hasalso demonstrated a variety of other interesting biological activities.It exerts antioxidant activity and is a potent scavenger of hydrogenperoxide. In addition, GEN has shown inhibitory abilities againstvarious enzymes involved in tumor development and growth such as proteintyrosine kinases, DNA topoisomerases, and protein kinase C (PKC). It hasalso shown to inhibit angiogenesis and induce apoptosis and cell cyclearrest in the G2-phase. While GEN is the most extensively studiedflavonoid for its versatile biological activities, it has been reportedto exert no or very weak aromatase inhibition.

Aromatase has been a particularly attractive target for inhibition inthe treatment of hormone-dependent breast cancer since the aromatizationis the last step in steroid biosynthesis and is the rate-limiting stepfor estrogen synthesis. In general, however, isoflavones are consideredless effective than flavones or flavonones in terms of aromataseinhibition. For this reason, to the best of our knowledge, there hasbeen no medicinal chemistry effort to develop isoflavones-basedaromatase inhibitors.

SUMMARY OF THE INVENTION

In one aspect, provided herein are compounds for treating or preventingcancer, particularly breast cancer in a subject. The compounds are newheteroaryl-containing isoflavones and pharmaceutical compositions thatcontain one of more of the heteroaryl-containing isoflavones asdescribed herein and derivatives thereof. It is preferred that thecompounds described herein are aromatase inhibitors. Theheteroaryl-containing isoflavones have the following base structure:

whereinX is selected from the group consisting of O, N, S, S(CH₂)_(n), whereinn=1-10; SO, and SO₂; and preferably, X is S or S(CH₂)_(n), wherein n=1,2, 3, and so forth;R₁ and R₂ can be the same or different and are selected from the groupconsisting of H, OH, OCH₃, OCH₂CH₃, OCH₂C₆H₅, NH₂, NHCH₃, N(CH₃)₂, CH₃,CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH, SCH₃,SCH₂CH₃, OCOCH₃, OCOC(CH₃)₃, OCOCH₂COOH, and CN; and

R₃ comprises a nitrogen-containing heterocyclic moiety. Preferably, R₃is a 5- or 6-membered ring containing 1-3 nitrogen atoms in the ring,though other sized rings and other numbers of nitrogen atoms in the ringmay be used. The ring may be selected from, but is not limited to, thegroup consisting of imidazole, triazole, pyrimidine, and pyridine. Thenitrogen-containing heterocyclic moiety may contain substituents on theheterocyclic ring. Those substituents may be selected from the groupconsisting of H, OH, OCH₃, OCH₂CH₃, NH₂, NHCH₃, N(CH₃)₂, NO₂, CH₃,CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃,CN and combinations thereof.

Some preferred compounds described herein include the followingstructures wherein R₁ and R₂ are as described above and R₃ is selectedfrom the following:

wherein X and R₁, R₂, and R₃ are as described above, and wherein X—R₃ isselected from:

Also provided is a method of using the compounds described herein in thetreatment of cancer. In one embodiment, the method is a method for thetreatment of breast cancer, particularly, but not limited tohormone-dependent breast cancer. The method comprises administering atherapeutically effective amount of a heteroaryl-containing isoflavoneof the present invention, or a derivative or pharmaceutically acceptablesalt or ester thereof, to a subject in need of treatment. Theheteroaryl-containing isoflavone compound can be administered inaccordance with conventional methods, and in doses similar to drugscurrently available for the treatment of breast cancer. Theheteroaryl-containing isoflavone compounds of present invention may alsobe used as part of a combination therapy. Also provided is a method forthe prevention of breast cancer in subjects who are susceptible todeveloping breast cancer, comprising administering a therapeuticallyeffective amount of a compound of a compound of the present invention,or a derivative or pharmaceutically acceptable salt or ester thereof.

Further provided is a method for the preparation of 2-substitutedisoflavones, the method comprising the steps of a) reactingdeoxybenzoins with a phase transfer catalyst to provide a2-(alkylthio)isoflavone; b) deprotecting the 2-(alkylthio)isoflavone;and c) applying selective debenzylation to form the final compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structures of several representativenonsteroidal aromatase inhibitors.

FIG. 2 shows Lineweaver-Burke plot and slope replot of compound 3j.

FIG. 3 shows two synthetic schemes used to prepare isoflavones.

DETAILED DESCRIPTION OF THE INVENTION

The role of endogenous estrogens in the development of hormone-dependentbreast cancer has been widely recognized, and inhibition of thebiosynthesis of estrogens has been on the most promising and logicaltherapeutic strategies for the disease. Among a number of enzymesinvolved in estrogen biosynthesis, aromatase is a particularlyattractive target for inhibition because aromatization is the final,rate-limiting step in estrogen biosynthesis; therefore, its blockadeshould not interfere with the production of other steroids. Aromataseinhibitors are typically classified by the chemical structures, namely,steroidal aromatase inhibitors and non-steroidal aromatase inhibitors.The nonsteroidal compounds are reversible inhibitors that compete withthe natural substrates, androstenedione and testosterone, for binding tothe active site of aromatase.

The present invention provides both new heteroaryl-containing isoflavonecompounds and methods of using those compounds. The compounds of thepresent invention encompass 2-(4-pyridylmethyl)thioisoflavones, as wellas salts, esters, and derivatives and related compounds. The presentinvention further encompasses derivatives of these compounds as well aspharmaceutical compositions that contain one of more of theheteroaryl-containing isoflavones of the present invention. Theheteroaryl-containing isoflavones have the following structure:

wherein

X is selected from the group consisting of O, N, S, and S(CH₂)_(n),wherein n=1-10; SO, and SO₂; and preferably, X is S, S(CH₂)_(n), whereinn=1, 2, 3, and so forth;

R₁ and R₂ can be the same or different and are selected from the groupconsisting of H, OH, OCH₃, OCH₂CH₃, OCH₂C₆H₅, NH₂, NHCH₃, N(CH₃)₂, CH₃,CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH, SCH₃,SCH₂CH₃, OCOCH₃, OCOC(CH₃)₃, OCOCH₂COOH, and CN;

R₃ comprises a nitrogen-containing heterocyclic moiety. Preferably, R₃is a 5- or 6-membered ring containing 1-3 nitrogen atoms in the ring,though other sized rings and other numbers of nitrogen atoms in the ringmay be used. The ring may be selected from, but is not limited to, thegroup consisting of imidazole, triazole, pyrimidine, and pyridine. Thenitrogen-containing heterocyclic moiety may contain substituents on theheterocyclic ring. Those substituents may be selected from the groupconsisting of H, OH, OCH₃, OCH₂CH₃, NH₂, NHCH₃, N(CH₃)₂, NO₂, CH₃,CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂, C(CH₃)₃, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃,CN and combinations thereof.

While the compounds of the present invention may work through anymechanism, it is preferred, though not required, that the compounds havearomatase inhibitory activity.

Some preferred compounds of the present invention include the followingwherein R₁ and R₂ are as described above and R₃ is selected from thefollowing:

wherein X and R₁, R₂, and R₃ are as described above, and wherein X—R₃ isselected from:

By “treating” is meant curing, ameliorating or tempering the severity ofthe cancer or the symptoms associated therewith. The terms “treating,”“treatment,” and “therapy” as used herein refer to curative therapy,prophylactic therapy, and preventative therapy.

“Preventing” or “prevention” means preventing the occurrence of thecancer, or tempering the severity of the cancer if it is developssubsequent to the administration of the instant compositions. Thispreventing the onset of a clinically evident unwanted cell proliferationaltogether or preventing the onset of a preclinically evident stage ofunwanted rapid cell proliferation in individuals at risk. Also intendedto be encompassed by this definition is the prevention of metastatis ofmalignant cells or to arrest or reverse the progression of malignantcells. This includes prophylactic treatment of those at risk ofdeveloping precancers and cancers.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of improvement in disease severity and the frequency of incidenceover treatment of each agent by itself, while avoiding adverse sideeffects typically associated with alternative therapies.

The term “subject” for purposes of treatment includes any human oranimal subject having a neoplasia, such as cancer or precancer. Formethods of prevention the subject is any human or animal subject, andpreferably is a human subject who is at risk of developing a cancer. Thesubject may be at risk due to exposure to carcinogenic agents, beinggenetically predisposed to disorders characterized by unwanted, rapidcell proliferation, and so on. Besides being useful for human treatment,the compounds of the present invention are also useful for veterinarytreatment of mammals, including companion animals and farm animals, suchas, but not limited to dogs, cats, horses, cows, sheep, and pigs.Preferably, subject means a human.

The term “derivative” is intended to encompass compounds which arestructurally related to the present invention or which possess thesubstantially equivalent activity to the parent heteroaryl-containingisoflavone compounds, as measured by the derivative's ability to inhibitactivity in an in vitro estrogen dependent cell proliferation assayusing human breast cells (such as MCF-7). By way of example, suchcompounds may include, but are not limited to, prodrugs thereof. Suchcompounds can be formed in vivo, such as by metabolic mechanisms.

Where the term alkyl is used, either alone or with other terms, such ashaloalkyl or alkylaryl, it includes C₁ to C₁₀ linear or branched alkylradicals, examples include methyl, ethyl, propyl, isopropyl, butyl,tert-butyl, and so forth. The term “haloalkyl” includes C₁ to C₁₀ linearor branched alkyl radicals substituted with one or more halo radicals.Some examples of haloalkyl radicals include trifluoromethyl,1,2-dichloroethyl, 3-bromopropyl, and so forth. The term “halo” includesradicals selected from F, Cl, Br, and I.

The term aryl, used alone or in combination with other terms such asalkylaryl, haloaryl, or haloalkylaryl, includes such aromatic radicalsas phenyl, biphenyl, and benzyl, as well as fused aryl radicals such asnaphthyl, anthryl, phenanthrenyl, fluorenyl, and indenyl on so forth.The term “aryl” also encompasses “heteroaryls,” which are aryls thathave carbon and one or more heteroatoms, such as O, N, or S in thearomatic ring. Examples of heteroaryls include indolyl, pyrrolyl, and soon. “Alkylaryl” or “arylalkyl” refers to alkyl-substituted aryl groupssuch as butylphenyl, propylphenyl, ethylphenyl, methylphenyl,3,5-dimethylphenyl, tert-butylphenyl and so forth.

The agents of the present invention may be administered orally,intravenously, intranasally, rectally, or by any means which delivers aneffective amount of the active agent to the tissue or site to betreated. It will be appreciated that different dosages may be requiredfor treating different disorders. An effective amount of an agent isthat amount which causes a statistically significant decrease inneoplastic cell count, growth, or size. Neoplastic disorders responsiveto the agents of the present invention include, but are not limited to,breast cancer.

The dosage form and amount can be readily established by reference toknown treatment or prophylactic regiments. The amount of therapeuticallyactive compound that is administered and the dosage regimen for treatinga disease condition with the compounds and/or compositions of thisinvention depends on a variety of factors, including the age, weight,sex, and medical condition of the subject, the severity of the disease,the route and frequency of administration, the particular compoundemployed, the location of the unwanted proliferating cells, as well asthe pharmacokinetic properties of the individual treated, and thus mayvary widely. The dosage will generally be lower if the compounds areadministered locally rather than systemically, and for prevention ratherthan for treatment. Such treatments may be administered as often asnecessary and for the period of time judged necessary by the treatingphysician. One of skill in the art will appreciate that the dosageregime or therapeutically effective amount of the inhibitor to beadministrated may need to be optimized for each individual. Thepharmaceutical compositions may contain active ingredient in the rangeof about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mgand most preferably between about 1 and 200 mg. A daily dose of about0.01 to 100 mg/kg body weight, preferably between about 0.1 and about 50mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day.

The active agents may be administered along with a pharmaceuticalcarrier and/or diluent. The agents of the present invention may also beadministered in combination with other agents, for example, inassociation with other chemotherapeutic or immunostimulating drugs ortherapeutic agents. Examples of pharmaceutical carriers or diluentsuseful in the present invention include any physiological bufferedmedium, i.e., about pH 7.0 to 7.4 comprising a suitable water solubleorganic carrier. Suitable water soluble organic carriers include, butare not limited to corn oil, dimethylsulfoxide, gelatin capsules, etc.

Also included in the family of heteroaryl-containing isoflavonecompounds are the pharmaceutically acceptable salts thereof. The phrase“pharmaceutically acceptable salts” connotes salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically acceptable. Also included in the family ofheteroaryl-containing isoflavone compounds are esters thereof. Esters ofthe heteroaryl-containing isoflavone compounds may be prepared byconventional methods known to those skilled in the art.

Suitable pharmaceutically acceptable acid addition salts ofheteroaryl-containing isoflavone compounds may be prepared from aninorganic acid or from an organic acid. Examples of such inorganic acidsare hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric,and phosphoric acid. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids, examples of whichinclude formic, acetic, propionic, succinic, glycolic, gluconic, lactic,malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic,p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic,methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, galactaric,and galacturonic acids.

Suitable pharmaceutically acceptable base addition salts ofheteroaryl-containing isoflavone compounds include metallic salts madefrom aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc.Alternatively, organic salts made from N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine(N-methylglucamine) and procaine may be used form base addition salts ofthe heteroaryl-containing isoflavone compounds. All of these salts maybe prepared by conventional means from the correspondingheteroaryl-containing isoflavone compounds by reacting, for example, theappropriate acid or base with the heteroaryl-containing isoflavonecompounds.

The phrase “adjunct therapy” (or “combination therapy”), in defining useof a compound of the present invention and one or more otherpharmaceutical agent, is intended to embrace administration of eachagent in a sequential manner in a regimen that will provide beneficialeffects of the drug combination, and is intended as well to embraceco-administration of these agents in a substantially simultaneousmanner, such as in a single formulation having a fixed ratio of theseactive agents, or in multiple, separate formulations for each agent.

There are large numbers of antineoplastic agents available in commercialuse, in clinical evaluation and in pre-clinical development, which couldbe selected for treatment of cancers or other neoplasias by combinationdrug chemotherapy. Such antineoplastic agents fall into several majorcategories, namely, antibiotic-type agents, alkylating agents,antimetabolite agents, hormonal agents, immunological agents,interferon-type agents and a category of miscellaneous agents.Alternatively, other anti-neoplastic agents, such as metallomatrixproteases inhibitors may be used. Suitable agents which may be used incombination therapy will be recognized by those of skill in the art.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are capsules, tablets, powders, granules or asuspension, with conventional additives such as lactose, mannitol, cornstarch or potato starch; with binders such as crystalline cellulose,cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators such as corn starch, potato starch or sodiumcarboxymethyl-cellulose; and with lubricants such as talc or magnesiumstearate. The active ingredient may also be administered by injection asa composition wherein, for example, saline, dextrose or water may beused as a suitable carrier.

For intravenous, intramuscular, subcutaneous, or intraperitonealadministration, the compound may be combined with a sterile aqueoussolution which is preferably isotonic with the blood of the recipient.Such formulations may be prepared by dissolving solid active ingredientin water containing physiologically compatible substances such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions to produce an aqueous solution, andrendering said solution sterile. The formulations may be present in unitor multi-dose containers such as sealed ampoules or vials.

Chemistry

We designed a library of 2,4′,7-trisubstituted isoflavones as shown inFIG. 1. The synthesis of the target compounds was carried out asoutlined in Scheme 1 and Scheme 2 (FIG. 3). The starting deoxybenzoins1a-c are commercially available or can be easily prepared by knownprocedures. The 4-hydroxyl group of each 1 was selectively protectedwith methyl or benzyl group under Mitsunobu reaction conditions to givemonoalkyl ethers 2a-e in excellent yields (Scheme 1). The treatment ofeach deoxybenzoin 2 was treated with carbon disulfide and an alkylhalide in a THF-aqueous NaOH solution in the presence of 10 mole % oftetrabutylammonium hydrogensulfate (n-Bu₄N.HSO) gave the corresponding2-(alkylthio)isoflavone 3 in good to excellent yields. Dealkylation ofselected compounds 3 was performed with boron tribromide indichloromethane yielding hydroxy compounds 4a-f (Scheme 2). All attemptsto prepare the 7-hydroxy-4′-methoxy analog 4g from 3k using typicaldebenzylation procedures (i.e. catalytic hydrogenation reactions withvarious hydrogen sources in the presence of palladium on carbon) failedpresumably due to catalyst poisoning of the sulfide group of 3k. The useof one equivalent of boron tribromide at a low temperature also provedto be inefficient; only providing a mixture of 3k, 4f, and 4g. However,we found that BF₃.OEt₂-Me₂S reagent is mild enough to achieve theselective removal of benzyl, leaving the 4′-methoxy group intact to give4g in a good yield. This reaction condition was originally reported as amild alternative debenzylation method in order to avoid undesirable1,4-conjugate addition of ethancthiol to substrates containing a Michaelacceptor.

Biological Evaluation

The aromatase assay was performed according to the modified method ofthe procedure previously reported by our laboratory, in which humanplacental microsomes were used as the aromatase source. IC₅₀ values ofthe compounds are shown in Table 1 and aminoglutethimide (AG) was usedas a reference. The IC₅₀ value of BCA was also determined in our assaysystem for comparison. In order to examine the mode of aromataseinhibition, kinetic studies for selected compounds (3j, 4f, and 4g) werealso performed, and their apparent K; values are listed in Table 2 withapparent K_(m) values and K_(l)/K_(m), ratios. Lineweaver-Burk plot ofcompound 3j, the most potent analog in this series, is shown in FIG. 2.

TABLE 1 Aromatase Activity of isoflavones 3a-k and 4a-g

Log IC₅₀ Cmpd. R₁ R₂ R₃ IC₅₀ (μM) (nM) (±S.E)^(a) 3a OMe OBn allyl >100— 3b OMe OBn benzyl >100 — 3c H OMe allyl >100 — 3d H OMe benzyl >100 —3e H OMe (4-pyridyl)methyl 1.6 3.21 ± 0.11 3f H OMe (3-pyridyl)methyl9.2 3.96 ± 0.16 3g H OMe (2-pyridyl)methyl >100 — 3h Me OMe(4-pyridyl)methyl 3.0 3.48 ± 0.05 3i OMe OMe (4-pyridyl)methyl 2.0 3.36± 0.30 3j H OBn (4-pyridyl)methyl 0.21 2.33 ± 0.03 3k OMe OBn(4-pyridyl)methyl 0.53 2.72 ± 0.11 4a OH OH allyl N.D.^(b) — 4b OH OHbenzyl N.D.^(b) — 4c H OH benzyl N.D.^(b) — 4d H OH (4-pyridyl)methyl0.61 2.79 ± 0.11 4e H OH (3-pyridyl)methyl 3.6 3.56 ± 0.06 4f OH OH(4-pyridyl)methyl 0.28 2.44 ± 0.07 4g OMe OH (4-pyridyl)methyl 0.22 2.34± 0.04 ^(c)AG 2.8 3.45 ± 0.05 BCA 34 4.53 ± 0.06 ^(a)IC₅₀ values werecalculated by a nonlinear regression analsys (GraphPad Prizm). Eachdose-response curve contained ten concentrations, each in triplicate.^(b)Not determined. ^(c)Aminoglutethimide.

TABLE 2 Enzyme kinetic parameters for selected isoflavones and referencecompounds Apparent K_(i) Apparent K_(m) Compound (μM) (±S.E) (μM) (±S.E)K_(i)/K_(m) 3j 0.22 ± 0.02 0.13 ± 0.01 1.69 4f 0.31 ± 0.02 0.11 ± 0.072.82 4g 0.26 ± 0.02 0.10 ± 0.02 2.60 AG 1.41 ± 0.10 0.09 ± 0.01 15.7 BCA12 ± 5^(a ) — — ^(a)From literatureResults and Discussion

Bioassay results showed that the analogs lacking a pyridyl group (3a-dand 4a-c) are less active than those containing a pyridine moiety. Itwas also obvious that the position of the nitrogen atom of pyridylmoiety affected the inhibitory activity. 4-Pyridyl analog 3e (IC₅₀=1.6μM) exerts-6-fold more potent inhibitory activity than 3-pyridyl analog3f (IC₅₀=9.2 μM). In the 7-hydroxy analogs; the 4-pyridyl analog 4d(IC₅₀=0.61 μM) is ˜6-fold more potent than 3-pyridyl analog 4e (IC₅₀=3.6μM). Although the precise mechanisms of action of these compounds arenot clear at present, the pyridyl moiety is thought to be involved incoordination with the heme iron of aromatase. Based on this hypothesis,it may be postulated that the nitrogen atom of the 4-pyridyl group maybe in the more favorable position to coordinate to the heme iron thanthat of the 3- or 2-pyridyl group.

In addition, the 7-hydroxy compounds, 4d (IC₅₀=0.61 μM) and 4e (IC₅₀=3.6μM), exhibit ˜3-fold greater inhibitory activities than their 7-methoxyanalogs, 3e (IC₅₀=1.6 μM) and 3g (IC₅₀=9.2 μM), respectively. Thisresult suggests that the presence of a hydrogen-bond donor at the7-position might provide a favorable interaction with the enzyme. Withregard to 4′-substituent, while the 4′-methyl analog 3h (IC₅₀=3.0 μM)appears less potent than the others, 4′-hydrogen analog 3e (IC₅₀=1.6 μM)shows a slightly higher activity than 4′-methoxy analog 3h (IC_(50=2.0)μM). However, the relatively small difference in their activities mayindicate that the effects of 4′-substitutent may not be critical for theactivity. Interestingly, this trend is not observed in the 7-hydroxyanalogs, 4g and 4d; i.e. in the presence of 7-hydroxy group, 4′-methoxyanalog 4g (IC₅₀=0.22 μM) is more potent than 4′-hydrogen analog 4d(IC₅₀=0.61 μM). Interestingly, the difference in their potency between4′-methoxy and 4′-hydrogen substituents in the 7-hydroxy analogs appearsto be greater than that observed in the 7-methoxy analogs. In addition,the potency of 4′,7-dihydroxy analog 4f (IC₅₀=0.28 μM) was comparable tothat of 7-hydroxy-4′-methoxy analog 4g (IC₅₀=0.22 μM), but ˜2-foldgreater than that of 7-hydroxy-4′-hydrogen analog 4d (IC₅₀=0.61 μM).This result suggests that the presence of a proton acceptor at the4′-position in the 7-hydroxy analogs may be beneficial for the aromataseinhibitory activity.

To our surprise, 7-benzyloxy analog 3k (IC₅₀=0.53 μM), originallyprepared as a precursor of compound 4g, showed a potent inhibitoryactivity. This result was unexpected because the compound was thought tobe too bulky to fit in the active site of the enzyme due to the benzylgroup. The 7-benzyloxy-4′-hydrogen analog 3j (IC₅₀=0.21 μM) exerts aenhanced potency over 7-benzyloxy-4′-methoxy analog 3k and ˜8-foldgreater than 4′-hydrogen-7-methoxy analog 3e (7-OBn versus 7-OMe). Also,3j is 13-fold and 162-fold more potent than AG and BCA, respectively, inour assay system in terms of IC₅₀ values.

In kinetic studies, compounds 3j, 4f, and 4g demonstrated typicalcompetitive type of inhibition in the Lineweaver-Burke plots (FIG. 2,data for 4f and 4g are not shown), suggesting that they may inhibitaromatase activity by competing with androstenedione for the substratebinding site of the enzyme. K_(i)/K_(m) ratios of compounds 3j, 4f, and4g were calculated as a relative inhibitory potency (Table 2), and thesame order of potency (3j>4g>4f) was observed as in the IC₅₀ studies. Asreflected by its K_(i)/K_(m) ratio, compound 3j is the most potent inthis series and is ˜10-fold more potent than AG. 3j also demonstrates50-fold enhancement in potency compared to our natural product lead,BCA.

Experimental Section

Unless otherwise noted, chemicals were commercially available and usedas received without further purification. Moisture sensitive reactionswere carried out under a dry argon atmosphere in flame-dried glassware.Solvents were distilled before use under argon. Tetrahydrofuran wasdistilled from sodium metal in the presence of benzophenone;dichloromethane was distilled from calcium hydride. Thin layerchromatography was performed on precoated silica gel F254 plates(Whatman). Silica gel column chromatography was performed using silicagel 60A (Merck, 230-400 Mesh). Melting points were determined in openglass capillaries using a Thomas Hoover apparatus and are uncorrected.Infrared spectra were recorded on a Nicola Protege 460 spectrometerusing KBr pellets. High-resolution electrospray ionization mass spectrawere obtained on the Micromass QTOF Electrospray mass spectrometer atThe Ohio State Chemical Instrumentation Center. All the NMR spectra wererecorded on a Bruker DPX 250, or Bruker DRx 400 model spectrometer ineither DMSO-d₆ or CDCl₃. Chemical shifts (δ) for ¹H NMR spectra arereported in parts per million to residual solvent protons. Chemicalshifts (δ) for ¹³C NMR spectra are reported in parts per millionrelative to residual solvent carbons.

General Procedure for the Preparation of 4-alkoxydeoxybenzoins (2a-e)

To a solution of 2-aryl-1-(2,4-dihydroxyphenyl)ethanone (31.0 mmol) andalcohol (32.55 mmol) in THF (150 mL) was added triphenylphosphine (8.538g, 32.55 mmol), followed by diisopropyl azodicarboxylate (6.41 mL, 32.55mmol) at 0° C., and the resulting yellow solution was stiffed at 0° C.for 10 min. The solvent was removed under reduced pressure, and the oilyresidue was directly purified by silica gel column chromatography(eluting with EtOAc:hexane, 1:4) and recrystallization (EtOAc andhexane) to yield desired product.

1-(2-Hydroxy-4-methoxyphenyl)-2-phenylethanone (2a)

Using the previous procedure and starting from1-(2,4-dihydroxyphenyl)-2-phenylethanone (7.08 g, 31.0 mmol) andmethanol (1.32 mL, 32.55 mmol), 6.60 g (88%) of the title compound wasobtained as a white solid: mp 87-88° C. (lit. 92° C.); IR (KBr) 1635,1620, 1589, 1437, 1350, 1291, 1230, 1206, 1127, 1021, 958, 802, 739,729, 551 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 12.71 (br s, 1H), 7.74 (d,J=8.7 Hz, 1H), 7.31-7.34 (m, 2H), 7.23-7.26 (m, 3H), 6.40-6.44 (m, 2H),4.20 (s, 2H), 3.81 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 202.38, 166.61,166.30, 134.82, 132.47, 129.76, 129.16, 127.50, 113.58, 108, 27, 101.44,56.00, 45.27.

1-(2-Hydroxy-4-methoxyphenyl)-2-(4-methylphenyl)ethanone (2b)

Using the previous procedure and starling from1-(2,4-dihydroxyphenyl)-2-(4-methylphenyl)ethanone (7.51 g, 31.0 mmol)and methanol (1.32 mL, 32.55 mmol), 7.38 g (93%) of the title compoundwas obtained as a white solid: mp (EtOAc/hexane) 71-72° C.; IR (KBr)1639, 1623, 1590, 1516, 1508, 1439, 1388, 1355, 1268, 1231, 1205, 1131,10.33, 1010, 957, 800, 780, 571, 504, 491 cm⁻¹; ¹H NMR (400 MHz, CDCl₃)δ 12.72 (br s, 1H), 7.73 (d, J=8.6 Hz, 1H), 7.24-7.14 (m, 4H), 6.43-6.40(m, 2H), 4.1.5 (s, 2H), 3.81 (s, 3H), 2.31 (s, 3H); ¹³C NMR (100 MHz,CDC₃) δ 202.63, 166.56, 166.28, 137.14, 132.47, 131.71, 129.87, 129.61,113.60, 108.20, 101.43, 55.98, 44.89, 21.48; HRMS calculated forC₁₆H₁₆NaO₁(M+Na)⁺ 279.0997; found 279.0989.

1-(2-Hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (2c)

Using the previous procedure and starting from1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)ethanone (8.0 g, 11.0 mmol)and methanol (1.32 mL, 32.55 mmol), 6.85 g (81%) of the title compoundwas obtained as a white solid: mp 101-102° C. (lit. 104° C.); IR (KBr)1637, 1611, 1513, 1459, 1346, 1301, 1237, 1174, 1148, 1026, 797, 787,626, 0.522 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 12.72 (br s, 1H), 7.73 (d,J=8.8 Hz, 1H), 7.17 (d, J=8.6 Hz. 2H), 6.86 (d, J=8.6 Hz, 2H), 6.40-6.44(m, 2H), 4.13 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H); ¹³C NMR (100 MHz,CDCl₃) δ 202.73, 166.55, 166.27, 159.07, 132.42, 130.78, 126.73, 114.61,113.53, 108.21, 101.42, 55.99, 55.67, 44.37.

1-[2-Hydroxy-4-(phenylmethoxy)phenyl]-2-phenylethanone (2d)

Using the previous procedure and starting from1-(2,4-dihydroxyphenyl)-2-phenylethanone (7.08 g, 31.0 mmol) and benzylalcohol (3.37 mL, 32.55 mmol), 8.62 g (87%) of the title compound wasobtained as a white solid: mp 106-108° C. (lit. 104-105° C.); IR (KBr)1620, 1572, 1500; 1389, 1352, 1270, 1230, 1192, 1134, 1000, 974, 830,760, 729, 697, 628, 561 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 12.69 (br s,1H), 7.48 (d, J=8.7 Hz, 1H), 7.25-7.39 (m, 10H), 6.49-6.51 (m, 2H), 5.07(s, 2H), 4.20 (s, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 202.41, 166.20,16.5.68, 136.22, 134.79, 132.53, 129.76, 129.17, 129.14, 128.76, 127.95,127.51, 113.77, 108.75, 102.47, 70.66, 45.28; HRMS calculated forC₂₁H₁₈NaO₃ (M+Na)⁺ 341.1154; found 341.1136.

1-[2-Hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone (2e)

Using the previous procedure and starting from1-(2,4-dihydroxyphenyl)-2-(4-methoxyphenyl)ethanone (8.0 g, 31.0 mmol)and benzyl alcohol (3.37 mL, 32.55 mmol), 10.23 g (95%) of the titlecompound was obtained as a white solid: mp 97-98° C. (lit. 93-95° C.);IR (KBr) 1635, 1611, 1512, 1496, 1387, 1351, 1289, 1227, 1173, 1131,1029, 994, 948, 842, 830, 791, 745, 725, 695, 535 cm⁻¹; ¹H NMR (250 MHz,CDCl₃) δ 12.73 (s, 1H), 7.75 (d, J=9.6 Hz, 1H), 7.33-7.40 (m, 5H), 7.18(d, J=8.5 Hz, 2H), 6.87 (d, J=8.5 Hz, 2H), 6.53-6.49 (m, 2H), 5.07 (s,2H), 4.14 (s, 2H), 3.78 (s, 3H); ¹³C NMR (69.3 MHz, CDCl₃) δ 202.77,166.19, 165.63, 159.10, 136.26, 132.51, 130.82, 129.15, 128.77, 127.97,126.71, 114.63, 113.73, 108.71, 102.48, 70.6.5, 55.69, 44.38; HRMScalculated for C₂₂H₂₀NaO₄ (M+Na)⁺ 371.1259; found 371.1265.

General Procedure for the Preparation of 2-Alkylthioisoflavones (3a-k)from 2′-hydroxydeoxybenzoins

To a stirred mixture of a deoxybenzoin (1 mmol), carbon disulfide (0.6mL, 10 mmol), alkyl halide (2.2 mmol), and tetrabutylammoniumhydrogensulfate (34 mg, 0.1 mmol) in THF (3 mL) and water (1 mL) wasslowly added 10 M solution of NaOH in water (1.2 mL, 12 mmol) at roomtemperature. A slight exothermic reaction and a color change of themixture were observed. The resulting mixture was vigorously stirred atroom temperature for several hours, and the product was extracted withethyl acetate (2×10 mL). The combined organic layer was washed withwater (10 mL) and then with brine (10 mL), dried over MgSO₄, andfiltered. The filtrate was concentrated under reduced pressure, and theresidue was purified by silica gel column chromatography (eluting withMeOH/CHCl₃ or EtOAc/hexane) and recrystallization (EtOAc/hexane) toyield desired product.

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one(3a)

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone(0.348 g, 1.0 mmol) as a starting deoxybenzoin and ally bromide (0.208mL, 2.4 mmol) as an alkyl halide, 0.396 g (92%) of the title compoundwas obtained as a white solid: mp 101-102° C.; IR (KBr) 1618, 1509,1438, 0.1363, 1342, 1291, 1247, 1176, 1152, 1101, 1030, 944, 821, 740,696 cm⁻¹; ¹H NMR (250 MHz, CDCl₃) δ 8.13 (d, J=8.8 Hz, 1H), 7.33-7.46(m, 5H), 7.25 (d, J=8.7 Hz, 2H), 7.02 (dd, J=8.9, 2.3 Hz, 1H), 6.95 (d,J=8.7 Hz, 2H), 6.89 (d, J=2.3 Hz, 1H), 5.87 (ddt. J=16.9, 10.0, 6.9 Hz,1H), 5.25 (dd, J=16.9, 1.2 Hz; 1H), 5.11-5.06 (m, 3H), 3.82 (s, 3H),3.69 (d, J=6.9 Hz, 2H); ¹³C NMR (62.9 MHz, CDCl₃) δ 174.34, 163.40,163.10, 159.93, 158.38, 136.17, 133.30, 132.30, 129.20, 128.84, 128.48,127.95, 124.64, 122.88, 119.22, 117.86, 114.96, 114.36, 101.29, 71.01,55.65, 34.59; HRMS calculated for C₂₆H₂₂NaO₁S (M+Na)+ 453.1137; found453.1123. Anal. (C₂₆H₂₂O₄S.0.3H₂O) C, H.

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one(3b)

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone(0.348 g, 1.0 mmol) as a starting deoxybenzoin and benzyl bromide (0.274mL, 2.3 mmol) as an alkyl halide, 0.427 g (89%) of the title compoundwas obtained as a white solid: mp 131-1.32° C.; IR (KBr) 1618, 1508,1438, 1364, 1247, 1176, 1029, 822, 697 cm⁻¹; ¹H NMR (250 MHz, CDCl₃) δ8.14 (d, J=8.9 Hz, 1H), 7.22-7.48 (m, 10H), 7.21 (d, J=8.7 Hz, 2H), 7.03(dd, J=8.9, 2.3 Hz, 1H), 6.93 (d, J=8.7 Hz, 2H), 6.89 (d, J=2.3 Hz, 1H),5.17 (s, 2H), 4.28 (s, 2H), 3.81 (s, 3H); ¹³C NMR (62.9 MHz, CDCl₃) δ174.33, 163.61, 163.09, 159.91, 158.38, 136.61, 136.17, 132.25, 129.33,129.25, 129.18, 128.88, 128.49, 128.15, 127.96, 124.52, 122.42, 117.82,115.03, 114.36, 101.25, 71.01, 55.65, 36.17; HRMS calculated forC₃₀H₂₄NaO₄S (M+Na)⁺ 503.1293; found 503.1258. Anal. (C₃₀H₂₄O₄S.0.1H₂O)C, H.

7-Methoxy-3-phenyl-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one (3c)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol)as a starting deoxybenzoin and ally bromide (0.190 mL, 2.2 mmol) as analkyl halide, 0.314 g (96%) of the title compound was obtained as awhite solid (Method B): mp 117-118° C.; IR (KBr) 1635, 1615, 1585, 1546,1503, 1435, 1373, 1345, 1252, 1197, 1108, 1017, 942, 922, 831, 753, 701,662 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J=8.9 Hz, 1H), 7.30-7.44(m, 5H), 6.96 (dd, J=8.9, 2.4 Hz, 1H), 6.81 (d, J=2.3 Hz, 1H), 5.83-5.94(m, 1H), 5.27 (dd, J=16.9, 1.2 Hz, 1H), 5.14 (dd, J=10.1, 0.8 Hz, 1H),3.91 (s, 3H), 3.70 (d, J=6.9 Hz, 2H); ¹³C NMR (62.9 MHz, CDCl₃) δ174.21, 164.12, 163.40, 158.50, 133.20, 132.61, 131.08, 128.82, 128.68,128.39, 123.35, 119.27, 117.69, 114.51, 100.13, 56.31, 34.54; HRMScalculated for C₁₉H₁₆NaO₃S (M+Na)⁺ 347.0718; found 347.0705. Anal.(C₁₉H₁₆O₃S.0.2H₂O) C, H.

7-Methoxy-3-phenyl-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (3d)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol)as a starting deoxybenzoin and benzyl bromide (0.262 mL, 2.2 mmol) as analkyl halide, 0.365 g (97%) of the title compound was obtained as awhite solid: mp 153-154° C.; IR (KBr) 1636, 1617, 1586, 1546, 1502,1438, 1373, 1341, 1252, 1205, 1106, 1016, 942, 831, 699, 661 cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ 8.12 (d, J=8.9 Hz, 1H), 7.22-7.41 (m, 10H), 6.95(dd, J=8.9, 2.4 Hz, 1H), 6.81 (d, J=2.3 Hz, 1H), 4.30 (s, 2H), 3.91 (s,3H); ¹³C NMR (62.9 MHz, CDCl₃) δ 174.22, 164.10, 163.54, 158.48, 136.51,132.52, 131.04, 129.32, 129.17, 128.81, 128.66, 128.39, 128.17, 122.97,117.70, 114.49, 100.19, 56.31, 36.17; HRMS calculated for C₂₃H₁₈NaOS(M+Na)⁺ 397.0874; found 397.0856. Anal. (C₂₃H₁₈O₃S.0.1H₂O) C, H.

7-Methoxy-3-phenyl-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (3e)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol)as a starting deoxybenzoin and 4-(bromomethyl)pyridine hydrobromide(0.557 g, 2.2 mmol) as an alkyl halide, 0.305 g (81%) of the titlecompound was obtained as a white solid: mp 136-137 C; IR (KBr) 1634,1622, 1600, 1549, 1497, 1433, 1369, 1257, 1200, 1098, 1067, 1013, 943,831, 775, 756, 703, 658, 570 cm+⁻¹; ¹H NMR (400 MHz. CDCl₃) δ 8.53 (dd,J=4.5, 1.5 Hz, 2H), 8.09 (d, J=8.9 Hz, 1H), 7.33-7.42 (m, 3H), 7.24-7.27(m, 4H), 6.93 (dd, J=8.9, 2.4 Hz, 1H), 6.70 (d, J=2.3 Hz, 1H), 4.21 (s,2H), 3.87 (s, 3H); ¹³C NMR (100 MHz. CDCl₃) δ 174.09, 164.21, 161.95,158.33, 150.45, 146.38, 132.22, 130.95, 128.89, 128.86, 128.46, 124.04,123.58, 117.59, 114.50, 100.16, 56.31, 34.79; HRMS calculated forC₂₂H₁₇NNaO₃S (M+Na)⁺ 398.0827; found 398.0818. Anal. (C₂₂H₁₇NO₃S.0.2H₂O)C, H, N.

7-Methoxy-3-phenyl-2-[(3-pyridylmethyl)thio]-4H-1-benzopyran-4-one (3f)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol)as a starting deoxybenzoin and 3-(bromomethyl)pyridine hydrobromide(0.557 g, 2.2 mmol) as an alkyl halide, 0.334 g (89%) of the titlecompound was obtained as a white solid: mp 151.5-152° C.; IR (KBr) 1635,1617, 1.585, 1547, 1503, 1438, 1427, 1373, 1344, 1253, 1203, 1108, 1017,943, 831, 754, 701, 662 cm⁻¹; ¹H NMR (400 MHz. CDCl₃) δ 8.62 (br s, 1H),8.49 (d, J=4.3 Hz, 1H), 8.10 (d, J=8.9 Hz, 1H), 7.69 (d, J=7.9 Hz, 1H),7.35-7.41 (m, 3H), 7.23-7.28 (m, 3H), 6.95 (dd, J=8.9, 2.1 Hz, 1H), 6.79(d, J=2.1 Hz, 1H), 4.27 (s, 2H), 3.91 (s, 3H); ¹³C NMR (100 MHz, CDCl₃)δ 174.11, 164.24, 162.30, 158.42, 150.24, 149.35, 136.81, 132.93,132.29, 130.96, 128.86, 128.80, 128.41, 124.11, 12.3.46, 117.60, 114.66,100.10, 56.34, 33.21; HRMS calculated for C₂₂H₁₇NNaO₃S (M+Na)⁺ 398.0827;found 398.0840. Anal (C₂₂H₁₇NO₃S1.0H₂O) C, H, N.

7-Methoxy-3-phenyl-2-[(2-pyridylmethyl)thio]-4H-1-benzopyran-4-one (3g)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-phenylethanone (0.242 g, 1.0 mmol)as a starting deoxybenzoin and 2-(Bromomethyl)pyridine hydrobromide(0.557 g, 2.2 mmol) as an alkyl halide, 0.345 g (92%) of the titlecompound was obtained as a white solid: mp 168.5-169° C.; IR (KBr) 1634,1617, 1586, 1546, 1502, 1431, 1373, 1344, 1252, 1202, 1153, 1106, 1016,943, 831, 782, 752, 698, 661 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.47(ddd, J=4.9, 1.7, 0.9 Hz, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.74 (dt, J=7.7,1.8 Hz, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.30-7.39 (m, 3H), 7.34 (ddd,J=7.6, 4.9, 0.9 Hz, 1H), 7.17-7.19 (m, 3H), 7.03 (dd, J=8.8, 2.4 Hz,1H), 4.54 (s, 2H), 3.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.33,164.34, 163.86, 158.54, 157.30, 150.20, 137.88, 133.14, 131.46, 129.03,128.86, 127.75, 124.04, 123.43, 122.40, 117.23, 115.46, 101.23, 57.02,37.39; HRMS calculated for C₂₂H₁₇NNaO₃S (M+Na)⁺ 398.0827; found398.0819. Anal. (C₂₂H₁₇NO₃S.0.1H₂O) C, H, N.

7-Methoxy-3-(4-methylphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one(3h)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methylphenyl)ethanone (0.256 g,1.0 mmol) as a starting deoxybenzoin and 4-(bromomethyl)pyridinehydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.335 g (86%) ofthe title compound was obtained as a white solid: mp 157-160° C.; IR(KBr) 1628, 1598, 1585, 1543, 1497, 1434, 1373, 1343, 1254, 1198, 1182,1099, 1016, 936, 837, 814 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.52 (d, J=5.6Hz, 2H), 8.08 (d, J=8.9 Hz, 1H), 7.26 (d, J=5.5 Hz, 2H), 7.21 (d, J=7.8Hz, 2H), 7.14 (d, J=7.9 Hz, 2H), 6.92 (dd, J=8.9, 2.1 Hz, 1H), 6.69 (d,J=2.0 Hz, 1H), 4.20 (s, 2H), 3.87 (s, 3H), 2.35 (s, 3H); ¹³C NMR (100MHz, CDCl₃) δ 174.20, 164.15, 161.79, 158.32, 150.46, 146.37, 138.72,130.75, 129.67, 129.17, 128.46, 124.05, 123.46, 117.58, 114.44, 100.14,56.29, 34.79, 21.85; HRMS calculated for C₂₃H₁₉NNaO₃S (M+Na)⁺ 412.0983;found 398.1004. Anal. (C₂₃H₁₉NO₃S.0.1H₂O) C, H, N.

7-Methoxy-3-(4-methoxyphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one(3i)

Using 1-(2-hydroxy-4-methoxyphenyl)-2-(4-methoxyphenyl)ethanone (0.272g, 1.0 mmol) as a starting deoxybenzoin and 4-(bromomethyl)pyridinehydrobromide (0.557 g, 2.2 mmol) as an alkyl halide, 0.332 g (82%) ofthe title compound was obtained as a white solid: mp 140-141° C.; IR(KBr) 1622, 1609, 1549, 1510, 1434, 1369, 1343, 1288, 1250, 1199, 1180,1099, 1024, 961, 945, 835, 821, 778 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 8.53(d, J=5.8 Hz, 2H), 8.08 (d, J=8.9 Hz, 1H), 7.27 (d, J=5.7 Hz, 2H), 7.18(d, J=8.6 Hz, 2H), 6.92-6.94 (m, 3H), 6.69 (d, J=2.1 Hz, 1H), 4.21 (s,2H), 3.88 (s, 3H), 3.80 (s. 3H); ¹³C NMR (100 MHz, CDCl₃) δ 174.29,164.14, 161.82, 160.05, 158.32, 150.42, 146.46, 132.16, 128.48, 124.24,124.06, 123.11, 117.57, 114.42, 114.40, 100.13, 56.29, 55.65, 34.82;HRMS calculated for C₂₃H₁₉NNaO₄S (M+Na)⁺ 428.0932; found 428.0949. Anal.(C₂₃H₁₉NO₄S.0.1H₂O) C, H, N.

3-Phenyl-7-(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one(3j)

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-phenylethanone (0.318 g,1.0 mmol) as a starting deoxybenzoin and 4-(bromomethyl)pyridinehydrobromide (0.557 g, 2.2 mmol) as an allyl halide, 0.386 g (86%) ofthe title compound was obtained as a pale yellow solid: mp 169-170° C.;IR (KBr) 1619, 1599, 1584, 1540, 1491, 1440, 1372, 1344, 1259, 1196,1157, 1099, 991, 943, 836, 819, 781, 747, 695 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 8.51 (dd, J=4.5, 1.6 Hz, 2H), 8.11 (d, J=8.9 Hz, 1H), 7.34-7.45(m, 8H), 7.72-7.26 (m, 4H), 7.03 (dd, J=8.9, 2.3 Hz, 1H), 6.78 (d, J=2.3Hz, 1H), 5.1.5 (s, 2H), 4.19 (s, 21-1). ¹³C NMR (100 MHz, CDCl₃) δ174.03, 163.22, 161.99, 158.23, 150.61, 146.23, 136.06, 132.19, 130.94,129.25, 128.90, 128.88, 128.57, 127.88, 123.94, 123.59, 117.80, 115.11,101.25, 71.03, 34.78; HRMS calculated for C₂₈H₂₁NNaO₃S (M+Na)⁺ 474.1140;found 474.1136. Anal. (C₂₈H₂₁NO₃S.0.1H₂O) C, H, N.

3-(4-Methoxyphenyl)-7-(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one(3k)

Using 1-[2-hydroxy-4-(phenylmethoxy)phenyl]-2-(4-methoxyphenyl)ethanone(0.348 g, 1.0 mmol) as a starting deoxybenzoin and4-(bromomethyl)pyridine hydrobromide (0.557 g, 2.2 mmol) as an alkylhalide, 0.402 g (84%) of the title compound was obtained as a whitesolid: mp 169.5-170.5° C.; IR (KBr) 1616, 1539, 1509, 1440, 1414, 1371,1342, 1294, 1251, 1197, 1173, 1156, 1100, 1029, 991, 943, 824, 735, 697,665 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (d, J=5.8 Hz, 2H), 7.87 (d,J=8.8 Hz, 1H), 7.35-7.49 (m, 7H), 7.30 (d, J=2.3 Hz, 1H), 7.08-7.12 (m,3H), 6.93 (d, J=8.7 Hz, 2H), 5.26 (s, 2H), 4.39 (s, 2H), 3.75 (s, 3H);¹³C NMR (100 MHz, DMSO-d₆) δ 173.39, 163.30, 162.91, 159.90, 158.31,150.68, 147.69, 136.95, 132.64, 129.46, 129.08, 128.82, 127.86, 124.79,124.70, 122.37, 117.32, 115.95, 114.55, 102.25, 70.95, 55.96, 34.03;HRMS calculated for C₂₉H₂₃NNaO₄S (M+Na)⁺ 504.1245; found 504.1238. Anal.(C₂₉H₂₃NO₄S.0.2H₂O) C, H, N.

General Procedure for Dealkylation using Boron Tribromide (4a-g)

To a stirred solution of 2-substituted7-alkoxy-3-aryl-4H-1-benzopyran-4-one (0.5 mmol) in CH₂Cl₂ (10 mL) wasslowly added a 1.0 M solution of BBr₃ (2.0 mL, 2 mmol) at 0° C., and theresulting suspension was allowed to warm to room temperature and stirredovernight. After cooling to 0° C., the reaction mixture was quenchedwith water and concentrated under reduced pressure. The residue wassuspended in a mixture of water and EtOAc, and the insoluble product wascollected by filtration. The filtrate was extracted with EtOAc twice(2×20 mL), and the combined organic layer was washed with brine, driedover MgSO₄, filtered, and concentrated under reduced pressure to giveadditional product. The combined solid was purified by silica gel columnchromatography (eluting with MeOH/CHCl₃) and/or directly applied torecrystallization.

7-Hydroxy-3-(4-hydroxyphenyl)-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one(4a)

Using the previous procedure and starting from3-(4-methoxyphenyl)-7-(phenylmethoxy)-2-[(propen-2-yl)thio]-4H-1-benzopyran-4-one(0.179 g, 0.416 mmol), 0.123 g (91%) of the title compound was obtainedas a pale yellow solid: mp 230-232° C. (decomposed); IR (KBr) 3231,1610, 1561, 1539, 1512, 1497, 1439, 1376, 1245, 1220, 1194, 1174, 1103,972, 949, 928, 846, 827, 809 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.72 (brs, 1H), 9.50 (br s, 1H), 7.81 (d, J=9.3 Hz, 1H), 6.99 (d, J=8.6 Hz, 2H),6.85-6.88 (m, 2H), 6.74 (d, J=8.6 Hz, 2H), 5.84 (ddt, J=16.9, 9.9, 6.9Hz, 1H), 5.27 (dd, J=16.9, 1.4 Hz, 1H), 5.09 (dd, J=9.9, 1.4 Hz, 1H),3.75 (d, J=6.9 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.61, 163.10,163.07, 158.51, 158.01, 134.75, 132.67, 128.07, 123.49, 122.50, 119.27,116.24, 115.83, 115.80, 102.70, 34.24; HRMS calculated for C₁₈H₁₄NaO₄S(M+Na)⁺ 349.0511; found 349.0529. Anal. (C₁₈H₁₄O₄S.0.1H₂O) C, H.

7-Hydroxy-3-(4-hydroxyphenyl)-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one(4b)

Using the previous procedure and starting from3-(4-methoxyphenyl)-7-(phenylmethox))-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one(0.147 g, 0.305 mmol), 0.106 g (92%) of the title compound was obtainedas a white solid: mp 261-264° C. (decomposed); IR (KBr) 3280, 1625,1605, 1592, 1508, 1459, 1375, 1246, 1228, 1189, 1174, 1111, 966, 948,843, 821, 694 cm⁻¹; ¹H NMR (400 MHz. DMSO-d₆) δ 10.75 (br s, 1H), 9.49(br s, 1H), 7.79 (d, J=8.7 Hz, 1H), 7.35-7.37 (m, 2H), 7.18-7.29 (m,0.3H), 6.94 (d, J=8.5 Hz, 2H), 6.91 (d, J=2.2 Hz, 1H), 6.86 (dd, J=8.7,2.2 Hz, 1H), 6.72 (d, J=8.5 Hz, 2H), 4.36 (s, 2H); ¹³C NMR (100 MHz,DMSO-d₆) δ 173.51, 163.25, 163.06, 158.48, 158.01, 138.18, 132.61,129.75, 129.40, 128.20, 128.06, 123.38, 121.96, 116.23, 115.86, 115.81,102.73, 35.38; HRMS calculated for C₂₂H₁₆NaO₄S (M+Na)⁺ 399.0667; found399.0656. Anal. (C₂₂H₁₆O₄S.0.5H₂O) C. H.

7-Hydroxy-3-phenyl-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (4c)

Using the previous procedure and starting from7-methoxy-3-phenyl-2-[(phenylmethyl)thio]-4H-1-benzopyran-4-one (0.223g, 0.596 mmol), 0.189 g (88%) of the title compound was obtained as awhite solid: mp 215-217° C.; IR (KBr) 3413, 1610, 1559, 1486, 1453,1375, 1269, 1250, 1219, 1194, 1105, 970, 947, 848, 699 cm⁻¹, ¹H NMR (400MHz, DMSO-d₆) δ 10.80 (br s, 1H), 7.81 (d, J=8.7 Hz, 1H), 7.20-7.37 (m,8H), 7.13-7.19 (m, 2H), 6.94 (d, J=2.2 Hz, 1H), 6.87 (dd, J=8.7, 2.2 Hz,1H), 4.38 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.31, 163.47, 163.20,158.54, 138.11, 133.26, 131.46, 129.76, 129.41, 128.99, 128.77, 128.24,128.08, 122.21, 116.20, 115.97, 102.79, 35.39; HRMS calculated forC₂₂H₁₆NaO₃S (M+Na)⁺ 383.0718; found 383.0710. Anal. (C₂₂H₁₆O₃S.0.4H₂O)C, H.

7-Hydroxy-3-phenyl-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one (4d)

Using the previous procedure and starting from7-methoxy-3-phenyl-2-[(4-pyidylmethyl)thio]-4H-1-benzopyran-4-one (0.165g, 0.439 mmol), 0.098 g (62%) of the title compound was obtained as apale yellow solid: mp 229-230° C. (decomposed); IR (KBr) 3427, 1617,1584, 1544, 1504, 1417, 1366, 1260, 1191, 1102, 1015, 969, 945, 843, 701cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.80 (br s, 1H), 8.48 (d, J=4.0 Hz,2H), 7.80 (d, J=8.4 Hz, 1H), 7.33-7.40 (m, 5H), 7.16 (d, J=7.0 Hz, 2H),6.86-6.88 (m, 2H), 4.38 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.28,163.19, 162.72, 158.48, 150.65, 147.82, 133.12, 131.45, 129.06, 128.87,128.09, 124.61, 122.50, 116.14, 116.01, 102.71, 34.05; HRMS calculatedfor C₂₁H₁₅NNaO₃S (M+Na)⁺ 384.0670; found 384.0667. Anal.(C₂₁H₁₅NO₃S.0.2H₂O) C, H, N.

7-Hydroxy-3-phenyl-2-[(3-pyridylmethyl)thio]-4H-1-benzopyran-4-one (4e)

Using the previous procedure and starting from7-methoxy-3-phenyl-2-[(3-pyridylmethyl)thio]-4H-1-benzopyran-4-one(0.181 g, 0.482 mmol), 0.082 g (47%) of the title compound was obtainedas a yellow solid: mp 194-197° C. (decomposed); IR (KBr) 3053, 1618,1584, 1542, 1502, 1462, 1366, 1268, 1219, 1187, 1102, 970, 946, 843,782, 754, 701 cm″; ¹H NMR (400 MHz, DMSO-d₆) S 10.82 (br s, 1H), 8.73(br s, 1H), 8.53 (d, J=4.4 Hz, 1H), 8.03 (d, J=7.8 Hz, 1H), 7.81 (d,J=8.7 Hz, 1H), 7.53 (dd, J=7.8, 5.1 Hz, 1H), 7.30-7.39 (m, 3H),7.13-7.15 (m, 2H), 6.95 (d, J=2.1 Hz, 1H), 6.89 (dd, J=8.7, 2.1 Hz, 1H),4.46 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.51, 163.12, 162.83,158.55, 148.07, 147.00, 140.30, 136.09, 132.92, 131.36, 129.15, 128.99,128.13, 125.64, 122.61, 116.10, 116.04, 102.79, 32.23; HRMS calculatedfor C₂₁H₁₅NNaO₃S (M+Na)⁺ 384.0670; found 384.0674. Anal.(C₂₁H₁₅NO₃S.0.3H₂O) C, H, N.

7-Hydroxy-3-(4-hydroxyphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one(4f)

Using the previous procedure and starting from3-(4-methoxyphenyl)-7-(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-benzopyran-4-one(0.173 g, 0.36 mmol), 0.117 g (86%) of the title compound was obtainedas a pale yellow solid: mp>240° C. (decomposed); IR (KBr) 3430, 1621,1607, 1560, 1513, 1499, 1369, 1263, 1234, 1194, 1171, 1107, 950, 807cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 10.74 (br s, 1H), 9.52 (br s, 1H),8.76 (d, J=6.4 Hz, 2H), 7.94 (d, J=6.4 Hz, 2H), 7.78 (d, J=9.0 Hz, 1H),6.97 (d, J=8.5 Hz, 2H), 6.86-6.88 (m, 2H), 6.76 (d, I=8.5 Hz, 2H), 4.58(s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 173.49, 163.15, 161.71, 158.42,158.17, 157.23, 144.66, 132.61, 128.07, 126.94, 123.07, 122.47, 116.14,115.91, 115.89, 102.83, 34.15; HRMS calculated for C₂₁H₁₅NNaO₄S (M+Na)⁺400.0619; found 400.0627. Anal. (C₂₁H₁₅NO₄S.0.1H₂O) C, H, N.

7-Hydroxy-3-(4-methoxyphenyl)-2-[(4-pyridylmethyl)thio]-4H-1-henzopyran-4-one(4g)

To a stirred suspension of3-(4-methoxyphenyl)-7-(phenylmethoxy)-2-[(4-pyridylmethyl)thio]-4H-1-henzopyran-4-one(0.200 g, 0.415 mmol) in Me₂S (3.0 mL) and CH₂Cl₂ (3.0 mL) was slowlyadded boron trifluoride diethyl etherate (1.52 mL, 12 mmol) at roomtemperature. The resulting yellow solution was vigorously stilled atroom temperature overnight. After cooling to 0° C., the reaction mixturewas quenched with water and concentrated under reduced pressure. Theresidue was suspended in a mixture of water and EtOAc, and the insolubleproduct was collected by filtration. The filtrate was extracted withEtOAc twice (2×2.5 mL), and the combined organic layer was washed withbrine, dried over MgSO₄, filtered, and concentrated under reducedpressure to give additional product. The combined solid was purified bysilica gel column chromatography (eluting with MeOH/CHCl₃) and thenrecrystallized from ethanol to give a white solid (0.136 g, 84%): mp197-200° C.; IR (KBr) 3400, 1623, 1606, 1561, 1512, 1455, 1363, 1293,1247, 1219, 1180, 1106, 1076, 1023, 1003, 968, 856, 822 cm⁻¹; ¹H NMR(400 MHz, DMSO-d₆) δ 10.76 (br s, 1H), 8.79 (d, J=6.4 Hz, 2H), 8.01 (d,J=6.5 Hz, 2H), 7.80 (d, J=8.5 Hz, I H), 7.11 (d, J=8.7 Hz, 2H), 6.94 (d,J=8.7 Hz, 2H), 6.86-6.89 (m, 2H), 4.56 (s, 2H), 3.73 (s, 3H); ¹³C NMR(100 MHz, DMSO-d₆) δ 173.45, 163.22, 161.74, 159.96, 158.48, 158.09,144.07, 132.68, 128.10, 127.18, 124.80, 122.28, 116.13, 115.98, 114.57,102.87, 56.00, 34.12; HRMS calculated for C₂₂H₁₈NO₄S (M+H)⁺ 392.0956;found 392.0962. Anal. (C₂₂H₁₇NOS.0.1H₂O) C, H, N.

Preparation of Human Placental Microsomes

Human Term Placentas were Processed immediately after delivery from TheOhio State University Hospitals at 4° C. The placenta was washed withnormal saline and connective and vascular tissue was removed. Microsomeswere prepared from the remaining tissue using the method described byKellis and Vickery. Microsomel suspensions were stored at −80° C. untilrequired.

Inhibition Study

Inhibition of human placental aromatase was determined by monitoring theamount of H₂O released as the enzyme converts[Iβ-³H]androst-4-ene-3,17-dione to estrone. Aromatase activity assayswere carried in 0.1 M potassium phosphate buffer (pH 7.0) with 5%propylene glycol. All samples contained a NADPH regenerating systemconsisting of 2.85 mM glucose-6-phosphate, 1.8 mM NADP′ and 1.5 units ofglucose-6-phosphate dehydrogenase (Sigma, St. Louis, Mo.). Samplescontained 100 nM androst-4-ene-3,17-dione (400,000-450,000 dpm).Reactions were initiated with the addition of 50 μg microsomal protein.Incubations were allowed to proceed for 15 minutes in a shaking waterbath at 37° C. Reactions were quenched by the addition of 2.0 mL ofchloroform. Samples were then vortexed and centrifuged for 5 minutes andthe aqueous layer was removed. The aqueous layer was subsequentlyextracted twice in the same manner with 2.0 mL chloroform. A 0.5 mLaliquot of the final aqueous layer was combined with 5 mL 3a70Bscintillation cocktail (Research Products International Corp., Mt.Prospect, Ill.) and the amount of radioactivity determined. Each samplewas run in triplicate and background values were determined withmicrosomal protein inactivated by boiling. Samples containing 50 μM (±)aminoglutethimide (Sigma, St. Louis, Mo.) were used a positive control.IC₅₀ sigmoidal dose-response data were analyzed with the Graphpad Prism(Version 3.0) program.

Kinetic Study

Enzyme kinetic studies of compounds 3j, 4f, and 4g were conducted toinvestigate the nature of aromatase inhibition. Michaelis-Menten enzymekinetic parameters were determined by varying the concentration ofandrost-4-ene-3,17-dione from 10 to 300 nM in the presence of a fixedconcentration of 0, 100, 500, and 2000 nM inhibitor. Assay conditionswere the same as those described in the IC₅₀ studies except reactionswere initiated by the addition of 15 lag microsomal protein. Analysis ofthe enzyme kinetic data was performed with the weighted linearregression analysis previously described by Cleland.

All documents referenced herein are incorporated by reference.

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations.

The invention claimed is:
 1. A method for treating breast cancer in asubject, comprising the step of administering to a subject that has beendiagnosed with breast cancer a therapeutically effective amount of anisoflavone of formula I

wherein X is selected from the group consisting of O, NH, S, SO, SO₂,and S(CH₂)_(n), wherein n=1-10; R₁ and R₂ can be the same or differentand are selected from the group consisting of H, OH, OCH₃, OCH₂CH₃,OCH₂C₆H₅, NH₂, NHCH₃, N(CH₃)₂, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂,C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, OCOCH₃, OCOC(CH₃)₃,OCOCH₂COOH, and CN; and R₃ is a 5- or 6-membered ring containing 1-3nitrogen atoms in the ring, the ring being unsubstituted or substitutedwith a member selected from the group consisting of H, OH, OCH₃,OCH₂CH₃, NH₂, NHCH₃, N(CH₃)₂, NO₂, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂,C(CH₃)₃, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, CN and combinations thereof.2. The method of claim 1 wherein X—R₃ is selected from the groupconsisting of:

wherein n is 0-3 and R₄ is selected from the group consisting of H, OH,OCH₃, OCH₂CH₃, NH₂, NHCH₃, N(CH₃)₂, NO₂, CH₃, CH₂CH₃, CH₂CH₂CH₃,CH(CH₃)₂, C(CH₃)₃, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, CN andcombinations thereof.
 3. The method of claim 1 wherein the breast canceris hormone-dependent breast cancer.
 4. A method for the synthesis of a2-substituted isoflavone, the method comprising the steps of: (a)reacting a deoxybenzoin with a phase transfer catalyst to provide a2-(alkylthio)isoflavone; (b) deprotecting the 2-(alkylthio)isoflavone;and (c) applying selective debenzylation to form an isoflavone of thefollowing formula I

wherein X is selected from the group consisting of O, NH, S, SO, SO₂,and S(CH₂)_(n), wherein n=1-10; R₁ and R₂ can be the same or differentand are selected from the group consisting of H, OH, OCH₃, OCH₂CH₃,OCH₂C₆H₅, NH₂, NHCH₃, N(CH₃)₂, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂,C(CH₃)₃, NO₂, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, OCOCH₃, OCOC(CH₃)₃,OCOCH₂COOH, and CN; and R₃ is a 5- or 6-membered ring containing 1-3nitrogen atoms in the ring, the ring being unsubstituted or substitutedwith a member selected from the group consisting of H, OH, OCH₃,OCH₂CH₃, NH₂, NHCH₃, N(CH₃)₂, NO₂, CH₃, CH₂CH₃, CH₂CH₂CH₃, CH(CH₃)₂,C(CH₃)₃, F, Cl, Br, CF₃, SH, SCH₃, SCH₂CH₃, CN and combinations thereof.5. The method of claim 1, wherein R₃ is a 4-pyridyl group.
 6. The methodof claim 1, wherein R₂ is a hydroxyl moiety.
 7. The method of claim 1,wherein R₁ is H, R₂ is OCH₂C₆H₅, and R₃ is 4-pyridyl.
 8. The method ofclaim 1, wherein the ring is selected from the group consisting ofunsubstituted or substituted imidazole, triazole, pyrimidine, andpyridine.
 9. The method of claim 8, wherein R₃ is imidazole, triazole,pyrimidine, or pyridine.
 10. The method of claim 4, wherein the ring isselected from the group consisting of unsubstituted or substitutedimidazole, triazole, pyrimidine, and pyridine.
 11. The method of claim10, wherein R₃ is imidazole, triazole, pyrimidine, or pyridine.